Immunological detection device and method for its preparation

ABSTRACT

An antigen-covered substrate is irradiated with ultraviolet light through a mask after which the exposed areas are rendered no longer antigenic. The unexposed areas retain their antigenic behavior and are available for the occurrence and detection of an immunological reaction.

BACKGROUND OF THE INVENTION

This invention relates to the detection of proteins by the utilizationof the phenomenum by which such proteins interact specifically eitherimmunologically or nonimmunologically.

Exemplary constructions of diagnostic devices for use in theimmunological detection of proteins are disclosed in U.S. Pat. Nos.3,926,564--Giaever, 4,011,308--Giaever, and 4,018,886--Giaever. Anarticle "Protein Deposition on Field-Emitter Tips and Its Removal By UVRadiation" by Panitz and Giaever [Surface Sci. 97, 25-42 (1980)]describes the removal of protein from the surface of a metal-coatedslide by the use of ultraviolet light in order to clean the surface.

The term "biological particle" is intended to encompass smaller proteins(e.g., plasma proteins, antigens, antibodies, lactins) and bodies ofproteinaceous material (e.g., viruses, bacteria, cells) capable ofstimulating antibody production, when injected into an animal and/orhaving the property of interacting specifically either immunologicallyor nonimmunologically.

Immunological reactions are highly specific biochemical reactions inwhich a first protein (e.g., an antigen) combines (links) with a secondprotein specific to the first protein (e.g., an antibody) to form animmunologically complexed protein. Immunological reactions taking placewithin a biological system, such as an animal or human being, are vitalin combating disease. In a biological system, the entry of a foreignprotein, i.e., an antigen, causes the biological system to produce theantibody proteins specific to the antigen by a process not fullyunderstood at this time. The antibody protein molecules have availablechemical combining or binding sites, which complement those of theantigen molecule, so that the antigen and antibody chemically link orbond to form an immunological complex protein.

Most antigens are proteins, or contain proteins as an essential part,whereas all antibodies are proteins. Proteins are large molecules ofhigh molecular weight, i.e., they are polymers consisting of chains ofvarious numbers of amino acids. A typical proteinaceous material willcomprise multiple entities (e.g., protein molecules, cells, etc.), whichdo not adhere to each other. Therefore, when a proteinaceous material isbrought into contact with a substrate, it deposites as a single layer.If the entities are molecular in size, this resulting single layer ismonomolecular; if the entities are larger, the layer will be a thickersingle layer. No other arbitrary protein will adhere to a depositedprotein layer. On the other hand, specifically reacting protein to aprotein adsorbed onto the substrate will immunologically bond thereto.

In accordance with the teachings in the above-cited patents, thisphenomenum is exploited to provide medical diagnostic apparatus in whicha substrate (e.g., a glass slide, the surface of which has been providedwith a layer of metal, such as indium) having a first layer of oneprotein adsorbed thereon is used to test suspected solutions for thepresence of the protein specific thereto (i.e. the protein specificallyreacting therewith). If the specifically reacting protein is present inthe solution, the substrate after exposure to the solution will have adouble protein layer thereon. If the specifically reacting protein isabsent from the solution, the slide after exposure to the solution willhave only the original layer thereon. Optical, electrical, chemical andtagged-detection means for distinguishing between the presence of doubleand single protein layers are known in the art and in the aforementionedpatents.

Because antibodies are produced by biological systems in response toinvasions thereof by foreign proteins, the detection of antibodies in abiological system is of medical diagnostic value in determining theantigens to which the system has been exposed. A typical example ofdiagnostic detection of antibodies is the detection of antibodies tosyphilis or gonorrhea in blood serum. Conversely, the detection ofcertain antigens in a biological system also has medical diagnosticvalue; examples of diagnostic detection of antigens include thedetection of HCG protein molecules in urine as a test for pregnancy, anddetection of hepatitus-associated-antigen (HAA) molecules in the bloodof prospective blood donors.

In order to perform such diagnostic tests, the appropriate protein ofthe immunologically reacting pair must be obtained. The only knownsource of an antibody protein is a living biological system. Moreparticularly, only vertebrates are known at this time to exhibitimmunological reactions to the introduction of a foreign protein. Forexample, many antibodies are found in the blood serum of animals andhuman beings which have been exposed to the corresponding antigens. Manyantigens, however, may be controllably produced in laboratory cultures.However, some antigens, for example, HAA molecules are at present likeantibodies, only obtainable from the higher living biological systems.

It is known in the immunological art that antibody molecules function asantigens when introduced into the system of a vertebrate to whom theyare foreign proteins. Accordingly, specifically reacting antibodies to agiven antibody may readily be produced in such vertebrate system.

While emphasis herein for the purposes of exemplification will be onimmunologically reactive biological particles (the simplest case beingthe antigen-antibody pair), it should be understood as explained at theonset that this invention is equally useful with sets of biologicalparticles that undergo forms of biological interaction other than theimmunologic reaction, the only criterion being that the particles mustbe mutually specific.

DESCRIPTION OF THE INVENTION

This invention is directed to a diagnostic device with improved readoutfor determining the presence or absence of select proteins in lowconcentration in a liquid sample and the method for its preparation. Thedevice comprises in combination a layer of biological particles coveringarea of one major surface of a substrate, the surface preferably beingmetallic, with the biological particles disposed within at least onefirst part of the area having the capability for interacting with otherbiological particles specific thereto and at least one second part ofthe area in which the biological particles do not have theaforementioned capability. The second part immediately adjoins (i.e.,borders upon) the first part in order to provide definition of the firstpart. Typically the first part of the overall area is shaped in apredetermined pattern to facilitate recognition of a positive test uponexposure to a liquid sample containing biological particles specific tothose disposed within the first part of the area.

In the method for constructing a typical diagnostic device embodyingthis invention, a glass slide having a coating of indium metal on itssurface is exposed to a solution containing antigen to cover the indiumsurface. After drying, this antigen layer is covered with a maskdefining one or more predetermined patterns, e.g. as openings throughthe mask, and this assembly is exposed to ultraviolet light for a periodof time sufficiently long to destroy the specificity of those biologicalparticles exposed to the ultraviolet light through the mask. Theexposure to ultraviolet light must be short enough (and the intensity ofthe source small enough) so that the protein layer remains intact.

Since most solids are opaque to ultraviolet light, the most feasiblemask construction is a solid mask with openings therethrough, e.g. holesor cut-outs in special shapes. However, a mask can be made of materialtransparent to ultraviolet light such as quartz with opaque portionsformed thereon.

By exposing the top surface of the diagnostic device to a solutioncontaining antibodies specific to the antigen, the non-irradiatedantigen pattern will pickup the specific antibody while the deactivated(radiated) antigen will not. This method of detection device preparationprovides a convenient way to set down discrete patterns and permits theuse of intricate patterns easily detectable by the unaided eye.

By having areas of irradiated biological particles bounded by the samebiological particles that have been rendered inactive, detection of thespecific reaction is enhanced, because of the increase in the opticaldensity from its initial value, where the specific protein adheres overthe predetermined pattern, which has retained its specificity. If theprotein to be detected is absent from the serum, the predeterminedpatterns remain invisible because both the areas which have retainedtheir specificity and those which have been rendered inactive have thesame optical density.

This method has the special advantage that it lends itself to moreaccurate characterization of faint images that could be due tonon-specific sticking of protein or to the presence of the selectprotein in very low concentration.

BRIEF DESCRIPTION OF THE DRAWING

The subject matter of the instant invention for which protection issought is presented as claims at the conclusion of the writtendescription of the invention set forth herein. The description setsforth the manner and process of making and using the invention and theaccompanying drawing forms part of the description schematicallyillustrating one embodiment. The views include:

FIG. 1 is a plan view of an exemplary mask construction;

FIG. 2 is a plan view of a metallized glass plate coated with a proteinlayer that has been irradiated with ultraviolet light through the maskof FIG. 1 (mask removed);

FIG. 3 is a sectional view taken on line 3--3 of FIG. 2;

FIG. 4 is a plan view of the slide of FIG. 2 after exposure tobiological particles specific to the active biological particles in thediscrete patterned areas;

FIG. 5 is a sectional view taken on line 5--5 of FIG. 4.

FIG. 6 is a plan view of a second embodiment of mask construction;

FIG. 7 is a plan view similar to FIG. 2 of a protein-coated metallizedglass irradiated using the mask of FIG. 6;

FIG. 8 is a sectional view taken on line 8--8 of FIG. 7;

FIG. 9 is a plan view of the slide of FIG. 7 after exposure tobiological particles specific to the active biological particles in thearea surrounding the discrete patterned areas, and

FIG. 10 is a sectional view taken on line 10--10 of FIG. 9.

MANNER AND PROCESS OF MAKING AND USING THE INVENTION

As used herein, terms such as "metallized surface" or "surface beingmetallic" encompass both surface areas of metal and surface areas ofmetal containing the oxide of one or more metals present. The substrateused herein is preferably glass slide with a surface thereof metallizedwith indium. However, suitable substrates may also be prepared frommetal, metallized plastic, or similar material although a method ofreadout other than the use of the unaided eye may be necessary.

Referring now to FIGS. 1 and 2 preparation of an exemplary diagnosticdevice according to this invention is as follows: A first layer 11 ofimmunologically reactive biological particles is deposited on the indiumlayer 12 adhered to glass slide 13. Coating 11 is usually applied bydipping the metal coated slide into a solution of the biologicalparticles (e.g. some specific antigen). After drying the slide, theimposition of discrete patterns on the coated slide is accomplished bythe use of mask 14 made of a material opaque to ultraviolet light havinga cut-out replica of the desired pattern, in this case the letter "G".

With the mask 14 in place over the surface of layer 11, this layer isirradiated with ultraviolet light through the mask. The time of exposureis relatively short. In the case of an ultraviolet light source suchHanovia type SH high pressure mercury arc lamp, the exposure time wouldbe about 3 minutes. The exposure period should be long enough to destroythe specificity of the biological particle(s) irradiated, but yet shortenough so that the biological particles will not be removed from thesurface by the exposure.

The exposed areas of coating 11 are eroded by the combined action of theultraviolet radiation and ozone so that the pattern of keying sites onthe biological particles that would be recognized by the select is areno longer present. The nature of this erosion mechanism is such that itis expected that in addition to applying to antigen-antibody couples andenzyme-substrate couples it should have general applicability tomutually specific biological particle couples.

After the irradiation step and subsequent removal of mask 14, the device15 (as shown in FIG. 2) will have discrete patterned areas 16 occupiedby non-irradiated biological particles and these patterns are surroundedby area in which the specificity of the protein layer has beendestroyed. These patterns 16 cannot be seen by visual inspection(because there is no change in optical density) and are, therefore,shown in the drawings as regions defined by dotted lines. The device 15is now ready for use.

Upon exposure of layer 11 to a sample of serum suspected of containingbiological particles specific to those comprising patterned areas 16 oneof two results will occur. If the specific biological particles are notpresent in the serum, spots 16 remain invisible, because no proteinswill attach and no contrasting area(s) of optical density is produced.If the specific biological particles are present, they will becomeattached to by the biological particles in patterned areas 16 to formlayers 17, but not by the surrounding previously irradiated area 18 asis shown in FIG. 4. The imposition of layers 17 over patterned areas 16increases the optical density in these regions and patterned areascoincident with areas 16 now become visible to the naked eye.

Although a letter-shaped pattern has been described to illustrate theinvention, more intricate patterns easily detectable by the eye may becreated using an appropriate mask. The mask material may, of course, betransparent, rather than opaque, to ultraviolet light.

A second embodiment shown in FIGS. 6-10 is exemplary of a particularlyeffective mode of detecting the difference between a positive readingfor a low concentration of the select protein and a negative readingcaused by the spurious presence of non-specific protein.

Mask 20 of material opaque to ultraviolet radiation has a large numberof very small holes (e.g. about 1 mm. in diameter) 21 in somepreselected pattern, such as closely spaced rows extending at rightangles to each other. After irradiation of a glass slide 22 (prepared asdescribed for slide 15 hereinabove) through mask 20, layer 11 will bepopulated by a very large number of very small invisible irradiatedareas 23. Only a portion of the irradiated areas are shown in thedrawings to simplify the illustration.

This arrangement is of particular advantage in the screening of largenumbers of samples to identify which one(s) contain the select protein.The screening is conveniently accomplished by applying a droplet 24 fromeach sample, each droplet covering several of the closely-spacedirradiated areas 23.

After an appropriate contact time, droplets 22 are washed off and slide22 is examined. Any positive readout such as region 26 will consist of alayer of the select biological particles covering all of the areaoccupied by droplet 24 except for the irradiated areas 23. The readoutis enhanced by the presence of several of the uncoated areas 23.

The embodiment of FIGS. 6-10 has been used to screen large numbers ofsamples of media from tissue cultures of hybridomas to determine whichhybridomas (if any) is the one productive of the sought-for monoclonalantibodies. In one instance, the objective was to locate the mediumcontaining monoclonal antibodies of aspartate aminotransferase. A slide22 (covered with a layer of aspartate aminotransferase) was preparedwith irradiated spots 23 in the form of 1 mm. diameter areas in rows 1mm. apart. Droplets 24 (about 4 mm. in diameter) of media from candidatetissue cultures were applied, about 20 droplets per slide.

Still another useful configuration of irradiated/non-irradiated areas aspart of a diagnostic device according to this invention is obtained bythe use of a screen (e.g. of metal) as the mask, preferably a screenwith very small (e.g. about 1 mm. square) openings.

What is claimed is:
 1. A method for making a diagnostic device fordetecting the presence or absence of select biological particles in aliquid sample comprising the steps of(1) contacting the surface of asubstrate with a layer of antigen particles specific for the detectionof the select biological particles, (2) contacting the antigen coatedsubstrate surface with a patterned mask, (3) irradiating the patternedmask-treated substrate composite with UV light for a period of timesufficient to destroy the specificity of irradiated antigen particlesfor the select biological particles, and (4) removing the mask.
 2. Adiagnostic device made in accordance with the method of claim
 1. 3. Thedevice of claim 2 where the substrate is an indium substrate.